Experiments detailed in this thesis investigated the bacterial response towards electrophiles and how this could be exploited as a novel target for antimicrobial chemotherapy. The principal conclusions are: (1) The root colonising bacterium <I>P. putida</I> does not appear to have a potassium efflux system similar to KefB and KefC. The exposure of log-phase <I>P. putida</I> cultures to sublethal concentrations of NEM resulted in a temporary viable non-culturable state, which could be suppressed by the presence of specific peptides in the recovery medium. Stationary phase cultures were more resistant to NEM, partially due to a faster metabolism, and did not undergo the temporary viable but non-culturable state. (2) Micotitre assays suitable for automated screening were developed to screen the Zeneca chemical library for activators of KefB and KefC and for activators and inhibitors of methylglyoxal synthase (MGS). (3) MGS was partially purified for <I>E. coli</I> and the N-terminal amino acid sequence was determined. This led to the identification of the gene encoding MGS, <I>mgsA</I>. An MGS over-expression vector and a chromosomal knock-out strain were constructed. (4) The over-expression of MGS resulted in an accumulation of MGS to approximately 25% of the total cell protein, corresponding to up to 900-fold enhanced MGS activity in crude extract. High level MGS expression had little effect on growth and viability of cells growth in LB, K<sub>10</sub> glucose or medium limited in phosphate. Severe growth inhibition was observed in cells over-expressing MGS in K<sub>10</sub> with either glycerol or xylose as the sole carbon source. (5) An MGS-deficient strain grew normally in LB, on glucose, glycerol, xylose, and in medium limited in phosphate or nitrogen. The MGS knock-out strain was severely inhibited during elevated xylose metabolism in the absence of methylglyoxal (MG) production. This suggests a prominent role for MGS in adaptation from "famine" (limited carbon) to "feast" (elevated carbon metabolism) conditions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:363684 |
| Date | January 1997 |
| Creators | Tötemeyer, Sabine |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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